1. Field of the Invention
The present invention relates to a fusion protein in which transferrin is peptide-bonded to a terminal of a granulocyte-colony stimulating factor (G-CSF) protein or a G-CSF mutant protein.
2. Description of the Related Art
Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein that stimulates survival, proliferation, differentiation, and functions of neutrophil and granulocyte progenitor cells and mature neutrophils.
The natural G-CSF which is now being used clinically is called ‘Filgrastim’, and is a recombinant protein composed of 175 amino acids originated from human amino acid sequence. This recombinant protein is expressed in E. coli and is not glycosylated unlike the natural type.
G-CSF is used as an anticancer adjuvant for the prevention of infectious complications caused by neutropenia accompanied by cancer treatment by stimulating neutrophil granulopoiesis. G-CSF demonstrates a beneficial clinical effect on cancer patients because it can reduce side effects of febrile neutropenia caused by chemo-therapy or radio-therapy for cancer treatment and thereby can reduce death rate by chemo-therapy for cancer treatment. G-CSF increases the number of hematopoietic progenitor cells and accordingly reduces the side effects above.
It is also known that G-CSF stimulates bone marrow stem cells to move ischemic heart and accelerates differentiation of the stem cells into vascular cells and cardiomyocytes by stimulating myocardial regeneration.
The recombinant human G-CSF (rhG-CSF) displays pharmacological effects only for a short time. Therefore, it has to be administered at least once a day to treat ischemic disease or to treat leukopenia caused by anticancer chemo-therapy or radio-therapy. If a substance having a long in vivo half-life is administered, the administration times necessary for relieving leukopenia would be reduced and as a result it could bring the effect of preventing infectious complications.
When Polyethylene glycol (PEG), the chemical polymer that is not degraded in vivo, is fused to the N-terminal of G-CSF, a substance called ‘Pegfilgrastim’ is produced. This substance is clinically used for the treatment of leukopenia. This substance has an increased in vivo half-life and displays a clinical effect while leukopenia continues even with the administration performed once or twice a week. However, in that case, a protein is fused with a chemical polymer by chemical reaction, and thus the problems of unsatisfactory fusion efficiency and complicated purification method can be caused.
Cysteine, the 17th amino acid of G-CSF is exposed on the protein surface as nonsulfated binding state. In neutral pH, the exposed cysteine is combined with adjacent G-CSF cysteine via sulfide bond and as a result it loses its activity. A G-CSF mutant wherein the 17th cysteine is substituted with serine displays increased stability in neutral pH, according to the previous reports.
Transferrin is the third most abundant protein in blood plasma, which serves to transport iron ions present in the blood to various tissues. Transferrin has a relatively long half-life of 8 days, which is shorter than that of albumin or immunoglobulin G. It enters the cell through the transferrin receptor on the surface of the cell and once it supplies iron ions, it is released to the outside of the cell in a state of binding with the receptor. Using these characteristics, transferrin has been used as a fusion partner to increase circulating half-life by combining the proteins with short half-lives of the prior art.
In this invention, threonine, the 116th amino acid of human G-CSF, was substituted with cysteine to induce sulfide bonding with the 17th cysteine of the original amino acid sequence of G-CSF. Sulfide bonding makes the protein structure more stable so that the protein can be resistant against proteases, that is the protein now has a protease resistance. The attempt to have a protease resistance through the construction of such a mutant protein is new and the effect thereof has not been reported yet.
There has been no reports of using the mutant protein of G-CSF wherein the 116th amino acid is replaced with cysteine for the fusion with transferrin to increase blood half-life of G-CSF.
The present inventors succeeded in making a fusion protein of transferrin and the mutant protein of human G-CSF wherein the 116th amino acid was replaced with cysteine and further confirmed that the fusion protein had significantly increased specific activity and blood stability, compared with the unfused original human G-CSF, leading to the completion of this invention.